Identifier

etd-11092004-134056

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is considered the etiologic agent of Kaposi’s sarcoma and several lymphoproliferative disorders. Recently, the full-length KSHV genome has been cloned into a bacterial artificial chromosome (BAC) and successfully recovered in 293 cells. The herpesviral glycoproteins are structural components of the KSHV particle and are thought to facilitate virus entry, egress and virus-induced cell fusion. Investigations, described herein, have focused on the genetic manipulation of the KSHV-BAC36 in order to address the role of K8.1 glycoprotein in virus entry and the role of the carboxyl tail a-helices of glycoprotein (gB) in virus-induced cell fusion. In addition, a panel of KSHV-glycoprotein mutants was constructed in order to address the specific role of each glycoprotein in viral entry, egress and virus-induced cell fusion. To further address the role of K8.1 in virus infectivity, a K8.1-null recombinant virus (BAC36-K8.1-null) was constructed by deletion of most of the K8.1 open reading frame and the insertion of a kanamycin resistance gene cassette within the K8.1 gene. Transfection of the mutant genome (BAC36-K8.1-null) DNAs into 293 cells produced infectious virions in the supernatants of transfected cells. Hence, these results clearly demonstrated that the K8.1 glycoprotein is not required for KSHV entry into 293 cells. In addition, two recombinant BAC36-derived genomes were constructed (via a two-step homologous recombination procedure in bacteria) specifying truncations that fully or partially truncated a predicted a-helical structure of the gB carboxyl terminus known to be involved in virus-induced cell fusion from studies with the herpes simplex virus type 1 (HSV-1) gB. Initial experiments suggested that disruption of the predicted a-helical structure gBtH2 enhanced virus-induced cell fusion. Furthermore, utilization of the pGET-Rec bacterial recombination system for insertional/deletional mutagenesis of the KSHV-BAC36 genome was successfully performed to produce the following KSHV-BAC36 null mutants: KSHV-BAC36-gB-null, KSHV-BAC36-gH-null, KSHV-BAC36-K8.1-null, KSHV-BAC36-gL-null and KSHV-BAC36-gM-null. The investigations herein have capitalized on the recent development of the KSHV-BAC36 clone in order to deliver targeted mutations to the KSHV genome.

Date

2004

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Konstantin G. Kousoulas

DOI

10.31390/gradschool_dissertations.682

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